Paired-End sequencing is a robust method for the study of gene expression and regulation. It links the 5′ terminal tags (˜18-20 bp each) of genomic DNA sequences or double stranded cDNA molecules directly to their corresponding 3′ terminal tags in the same order and orientation for high throughput sequencing. In practice, the Paired-End approach has been routinely used as a wetlab procedure for making libraries either from double-stranded cDNA molecules, or genomic DNA fragments enriched by chromatin immunoprecipitation (ChIP). The biological meanings of the transcriptomic or genomic sequences are subsequently unraveled by sequencing and bioinformatics analyses. Some applications of this approach were demonstrated in previous work for the development of the Paired-End Ditagging (PET) technology.
Next-veneration sequencers, e.g., SOLiD™ 3 sequencers manufactured by Applied Biosystems (AB), Solexa sequencers by Illumina, and 454 sequencers by Roche, have incorporated the Paired-End sequencing as an intrinsic feature. Currently, the SOLiD™ 3 system runs on three types of libraries, namely fragment library, Mate-Paired (MP) library, and barcoded fragment library (See SOLiD™ 3 System Library Preparation Guide, page 1, which is herein incorporated by reference in its entirety). The construction of a Mate-Paired or a barcoded fragment library is more complex than that of a fragment library, and the cost for sequencing is also almost twice as much. Thus, a design to bypass the current protocol of making Mate-Paired library constructs is strongly desirable and would be beneficial for the scientific community.
Therefore, a heretofore unaddressed need exists in the art to address the aforementioned deficiencies and inadequacies, especially in connection with development of a simplified protocol for making Paired-End library constructs that can lower the cost and allow high throughput sequencing.